Bivalent Bispecific Antibodies Research Articles

Deciphering integration loci of CHO manufacturing cell lines using long read nanopore sequencing

Despite advances in genetic characterization of Chinese hamster ovary (CHO) cell lines regarding identification of integration sites using next generation sequencing, e.g. targeted locus amplification sequencing (TLA-seq), the concatemer structure of the integrated vectors remains elusive. Here, the entire integration locus of two CHO manufacturing cell lines was reconstructed combining CRISPR/Cas9 target enrichment, nanopore sequencing and the Canu de novo assembly pipeline. An IgG producing CHO cell line integrated 3 vector copies, which were near full-length and contained all relevant vector elements such as transgenes and their promoters on each of the vector copies. In contrast, a second CHO cell line producing a bivalent bispecific antibody integrated 7 highly fragmented vector copies in different orientations leading to head-to-head and tail-to-tail fusions. The size of the vector fragments ranged from 3.0 to 11.4 kbp each carrying 1–3 transgenes. The breakpoints of the genome-vector and vector-vector junctions were validated using Sanger sequencing and Southern blotting. A comparison to TLA-seq data confirmed the genomic breakpoints, but most of the breakpoints of the vector-vector fusions were missed by TLA-seq. For the first time, the complete transgene locus of CHO manufacturing cell lines could be deciphered. Strikingly, the application of the nanopore long-read sequencing technology led to novel insights into the complexity of genomic transgene integrations of CHO manufacturing cell lines generated via random integration.

Abstract 2891: Enhanced anti-tumor responses with a novel dual pMHC T-cell engager bispecific antibody

Abstract Intracellular tumor antigens presented as peptides on MHC (pMHC) class I molecules are attractive targets for more tumor-selective immunotherapeutic approaches with promising data already emerging from clinical trials. pMHCs have been targeted by TCR-engineered T cells or soluble recombinant T-cell receptors (TCRs) fused to an anti-CD3 fragment. Naturally occurring cancer-reactive TCRs have weak affinity and require substantial affinity enhancements for their cognate pMHC. However, the outcome of this process is difficult to predict and bears the risk for off-target cross reactivities in normal tissues, which may lead to severe adverse events in the clinic. Here, we describe a highly potent dual pMHC T-cell engager (TCE) antibody format with high specificity towards tumor-specific pMHCs utilizing an HLA-A*02:01 restricted MAGE-A4 epitope to validate the concept. A series of monovalent and bivalent antibody constructs composed of anti-MAGE-A4 binding arms, ranging in affinities from 30 nM to 100 pM, were fused to an anti-CD3 Fab fragment with lower affinity compared to that commonly used for TCR-fusions. The different antibody constructs were evaluated for selective killing of MAGE-A4/HLA-A*02 positive human U2OS osteosarcoma and A375 melanoma cancer cells versus a panel of different MAGE-A4-negative/HLA-A*02-positive human cell lines. Bivalent bispecific antibody variants mediated a 7-fold greater degree of cancer cell killing and similarly increased T cell activation compared to their monovalent bispecific counterparts. EC50 values ranged as low as single digit picomolar, while the overall cross reactivity against MAGE-A4-negative/HLA-A*02-positive cells was not substantially affected. These results prove that dual targeting of pMHCs on cancer cells provides selective and efficient T cell-mediated target cell killing and T cell activation, even at very low levels of pMHC on the cell surface, highlighting the pivotal roles played by the affinity of the individual arms, valency, and epitope densities. We further analyzed the dual pMHC TCE for potential off-target effects by recognition of similar and physiologically relevant non-MAGE-A4 peptides. T2 cells pulsed with similar peptides and co-cultured with PBMC effector cells showed no significant T cell activation or IFNg release in the presence of the dual pMHC TCE in comparison to MAGE-A4 peptide-pulsed T2 cells. Finally, we compared the dual pMHC TCE against a recombinant TCR fused to an anti-CD3 scFv, a construct that is currently in clinical development. Interestingly, the dual pMHC TCE resulted in a 3-fold more potent cancer cell killing while having significantly lower effect on cytokine production. In conclusion, dual pMHC targeting with TCE antibodies is an attractive alternative to soluble affinity-enhanced TCR-based cancer immunotherapies as they facilitate potent tumor targeting without the need for extensive affinity enhancements. Citation Format: Stephanie Jungmichel, Fabian Scheifele, Anna Sobieraj, Severin Wendelspiess, Thomas Schleier, Philip Knobel, Camilla Winnewisser, Melissa Vrohlings, Philipp Richle, Hannes Merten, Jacqueline Blunschi, Christian Leisner, Leonardo Borras. Enhanced anti-tumor responses with a novel dual pMHC T-cell engager bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2891.

Abstract 5605: BH3120: A novel bispecific antibody targeting 4-1BB and PD-L1 with well balanced efficacy and safety profiles

Abstract 4-1BB (CD137, TNFSF9) is a promising co-stimulatory signaling mediator of T cells and NK cells, and agonistic monoclonal antibodies targeting 4-1BB are under clinical investigations aiming to observe sufficient and prolonged anti-tumor efficacy. However, these trials have resulted in limited efficacy or safety, and different bispecific approaches are being explored to improve the profiles. BH3120, a bivalent bispecific antibody generated by Pentambody(TM) platform targeting 4-1BB and PD-L1 simultaneously, demonstrates strong and prolonged antitumor efficacy as monotherapy in in vivo studies, and favorable safety profiles up to 200mg/kg/dose in non-human primates. Moreover, combination of BH3120 with an immune checkpoint inhibitor shows synergistically enhanced anti-tumor efficacy. Supported by these results, IND enabling studies of BH3120 are on-going and clinical evaluations are planned from later in 2022. Citation Format: Jiangcheng Xu, Yang Liu, Jing Wang, Fei Fan, Haitao Gu, Lanxin Zhang, Jun Wang, Ding Song, Ailong Guo, Hongjuan Zhang, Kaixuan Su, Jiaojiao Su, Lijuan Li, Jingmei Cai, Aihong Zhang, Jiawang Liu, Kyoungwoo Lee. BH3120: A novel bispecific antibody targeting 4-1BB and PD-L1 with well balanced efficacy and safety profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5605.

Role of configurational flexibility on the adsorption kinetics of bivalent bispecific antibodies on porous cation exchange resins

The adsorption kinetics of a monoclonal antibody (mAb) used as a reference and of bivalent bispecific antibodies (BiSAb) on a macroporous cation exchanger is studied experimentally by examining the transient patterns of bound protein within the particles using confocal microscopy for a range of protein concentrations, buffer concentrations and pH, and temperatures. The mAb adsorption kinetics is controlled by pore diffusion and conforms to the classical shrinking core model. While the mAb adsorption rate increases with temperature, the ratio of effective and free solution diffusivity, De /D0, remains constant and has a value of 0.20. The BiSAb's structure is comprised of scFv domains that are genetically fused to a framework IgG through flexible peptide linkers which results in conformational flexibility leading to multiple binding forms with varying affinity for the adsorbent surface. As a result, adsorption of the BiSAbs shows complex patterns of total bound protein within the particles. These BiSAb adsorption patterns are influenced by buffer ionic strength, pH, and temperature in unique ways. Sharper intraparticle profiles are observed for conditions where the binding strength is greater (lower buffer concentration and/or pH) or when the protein is chemically crosslinked to restrict configurational flexibility. Temperature affects the BiSAb pore diffusivity as well as the interconversion kinetics. While the effects of temperature on BiSAb transport are also described by a constant De /D0 = 0.15, the temperature also affects the rate of interconversion between binding forms leading to faster equilibration at higher temperatures. A phenomenological model indicates that the interplay of pore diffusion and adsorption with the kinetically limited interconversion between binding forms is responsible for the experimental trends.

Chromatographic and adsorptive behavior of a bivalent bispecific antibody and associated fragments

The elution and adsorptive behavior of a bivalent bispecific antibody (BiSAb), comprising an IgG1 framework with a scFv domain genetically fused to each heavy chain C-terminus via flexible linkers, and of two associated fragments were studied on two cation exchange chromatography media – ProPac WCX-10, which is pellicular and suitable for analytical use, and Nuvia HR-S, which is macroporous and suitable for preparative and process scale uses. Both fragments were identified by MS as missing one of the two scFv domains and its flexible linker, but one of them also contains an additional C-terminal lysine. The separation of these fragments on both resins occurs as a result of differences in non-specific ligand-protein interactions that are modulated by the salt concentration. For the ProPac WCX-10 column, complex, multipeak elution behaviors are observed, since, as a result of the linker flexibility, both the intact molecule and the fragments appear to exist in multiple binding configurations with each scFv domains either collapsed onto the IgG framework or extended away from it. With a residence time of 2.5 min and at 21 °C, two peak elution is observed for the fragments which contain a single linked scFv and three peak elution for the intact molecule which contains two linked scFvs. This behavior is affected by residence time, temperature, and hold time. Increasing the residence time to 25 min or increasing temperature to 40°C results in elution of a single, merged peak for each of the protein species. For Nuvia HR-S, the broader peaks, obtained as a result of mass transfer limitations, tend to obscure the multipeak elution behavior. Nevertheless, even for this resin, the effects of configurational flexibility are still manifested at the single-particle scale and affect the evolution of the patterns of protein binding within individual resin particles as evident from confocal microscopy observations.

Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation.

Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme-induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single-chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv-fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody-like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.

Chromatographic behavior of bivalent bispecific antibodies on hydrophobic interaction chromatography columns

The elution behavior of bivalent bispecific antibodies (BiSAb) comprising an immunoglobulin G framework genetically fused to a pair of single chain variable fragments (scFvs) was studied on hydrophobic interaction chromatography (HIC) columns using ammonium sulfate gradients. Each of the BiSAb molecules studied exhibited a three-peak elution behavior regardless of the location of scFv attachment to the framework IgG. Collecting and re-injecting each of the isolated peaks and eluting with the same gradient resulted in the same three-peak profile indicating that the behavior is reversible. Analogous behavior was observed for HIC resins with different functional ligands, matrix structures, and particle sizes. Residence time, operating temperature, and hold time were shown to affect the elution behavior. While three peaks were obtained at short residence times and room temperature, residence times longer than about 27 min or operating at 45 °C resulted in a single merged peak indicating that the underlying mechanism occurs on time scales comparable to that of chromatographic separation. Holding the protein on the resins prior to elution enriched the late eluting peak indicating that multiple binding states formed on the chromatographic surface are responsible for this behavior. Tryptophan auto-fluorescence measurements show that stronger binding forms have increased solvent exposure indicating that surface-catalyzed conformational changes play a role. A model was developed to describe the interplay of chromatographic separation and slow conformational changes.

Chromatographic behavior of bivalent bispecific antibodies on cation exchange columns. I. Experimental observations and phenomenological model

The cation exchange chromatographic behavior of three homologous bivalent bispecific antibodies (BiSAb) is characterized for two different resins, Source 15S and ProPac WCX-10, having different base matrix, particle size, pore structure, and ligand chemistry. For both resins, elution with a salt gradient results in multiple peaks for each of the three BiSAb molecules at short residence times. The peaks gradually merge into two peaks and then into one peak eluting at intermediate salt concentrations when the residence time is gradually increased. Re-injecting fractions of each the individual peaks obtained at short residence time results in nearly the same multiple peak elution pattern. This behavior, which is contrary to the behavior normally encountered in ion exchange chromatography, appears to be related to the reversible, surface -catalyzed interconversion between different conformational states of each BiSAb that interact with different strength with the chromatographic surface. This behavior is qualitatively independent of pH in the range 5–8.5, protein load in the range 0.06–5.0 mg/ml, and gradient slope, and is not associated with the formation of aggregates. Gradually increasing temperature, however, reduces the multiple peak behavior eventually resulting into a single peak at 55 °C A phenomenological model is developed that predicts the experimental behavior over a broad range of conditions using fitted rate and equilibrium constants.

Chromatographic behavior of bivalent bispecific antibodies on cation exchange columns. II. Biomolecular perspectives

In Part I of this work we determined the experimental cation exchange behavior of bivalent bispecific antibodies (BiSAb) comprising a pair of single chain variable fragment (scFv) domains flexibly linked to a framework immunoglobulin G (IgG), which exhibit a complex, three-peak elution pattern dependent on the residence time. A phenomenological model was developed assuming that the BiSAb molecules exist in multiple configurations that interact differently with the resin surface and interconvert at finite rates. In Part II of this work we provide relevant biomolecular perspectives that shed light on the underlying mechanisms. Firstly, we show that crosslinking the BiSAb molecules with a bifunctional reagent, which limits conformational flexibility, suppresses multiple peak elution. Secondly, we show that of the fragments obtained by enzymatic digestion of the BiSAb molecules only those that exhibit a pair of scFv domains show three-peak elution, while only two peaks are observed if a single scFv is present. Thirdly, we analyze the roles of electrostatic and hydrophobic surface properties of the BiSAb domains, identifying regions that are likely responsible for inter-domain and protein-surface interactions. The results demonstrate that the complex elution behavior catalyzed by the combination of surface charge and hydrophobicity of the stationary phase is associated with outstretched and collapsed configurations of the scFv domains relative to the framework IgG.

Abstract 2775: A novel immunostimulatory OX40/PD-L1 bivalent bispecific antibody (MEDI1109) for the treatment of patients with cancer

Abstract Targeted immunotherapy utilizing agonist antibodies (Ab) to OX40 shows great promise in preclinical mouse models alone or in combination with various therapies including PD1/PD-L1 antagonist Abs. Ab-mediated signaling through OX40 requires higher-order clustering by Fcγ receptors to co-stimulate antitumor-specific T cells and attenuate regulatory T cell (Treg) immunosuppression, leading to antitumor activity. To date, OX40 agonists are well-tolerated but show limited activity alone or in combination with checkpoint blockade in clinical trials. We hypothesize that the antitumor activity of OX40 agonists in patients may be limited by insufficient intratumoral exposure and an inadequate amount of clustering by Fcγ receptors within the tumor microenvironment. To overcome these limitations, we engineered MEDI1109, a bivalent bispecific Ab composed of an OX40 agonist Ab molecularly fused to two single-chain variable fragments derived from a PD-L1 antagonist. The resulting bispecific Ab retained the functional properties of each parent Ab that included binding and agonizing OX40, binding and full blockage of the PD-L1/PD-1 pathway, depletion of OX40+ Tregs and overcoming Treg suppression. Emerging data suggest that the bispecific Ab also drives novel biology including (1) increased OX40 agonism through clustering by either PD-L1 and Fcγ receptors, (2) improved intratumoral exposure through PD-L1 targeting, (3) elimination of PD-L1 positive tumor cells by NK cell mediated antibody-dependent cellular cytotoxicity and (4) enhanced antitumor immunity through concurrent PD-L1/PD-1 blockade on tumor cells and OX40 co-stimulation of antitumor T cells. Intravenous administration of OX40/PD-L1 bispecific Abs to cynomolgus monkeys resulted in the proliferation of peripheral blood CD4+ and CD8+ total memory T-cell populations, and in the reduction of soluble PD-L1 levels in the plasma. These results demonstrate that MEDI1109 is a unique and potent immune modulating agent and may be utilized to enhance antitumor immunity in patients with cancer. Citation Format: Michael D. Oberst, Srinath Kasturirangan, Clifford Sachs, Catherine Auge, James Moynihan, Raymond Rothstein, James Hair, Francis Neal, Srinivas Mamidi, Shino Hanabuchi, Amanda Watkins, Yanan Zheng, Kim Rosenthal, Daniel J. Freeman, Scott Hammond. A novel immunostimulatory OX40/PD-L1 bivalent bispecific antibody (MEDI1109) for the treatment of patients with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2775.

Abstract 2643: A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers

Abstract To harness the cytotoxic capacity of T cells for the treatment of EGFRvIII+ cancers, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 (or CD16A) and EGFRvIII, the EGFRvIII/CD3 (and EGFRvIII/CD16A) RECRUIT-TandAb. EGFR dysregulation has been linked to numerous cancers, and both small molecules and EGFR targeting antibodies have successfully reached the clinic. The deletion variant III (EGFRvIII), the most common mutant form of EGFR is expressed exclusively in cancer tissues contributing to oncogenic transformation. The restricted EGFRvIII expression on various solid tumor types provides an opportunity to develop cytotoxic antibodies that solely target cancer, sparing normal tissues, and substantially reduce the side effects associated with EGFR therapy. Using phage display libraries, we identified scFvs that selectively bound to the mutated and not the native form of EGFR. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies; hence we engineered a panel of bispecific EGFRvIII/CD3 TandAbs, capable of T cell-recruitment, with a broad range of binding and cytotoxic properties. The TandAbs' binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. They exhibited exquisite specificity towards EGFRvIII in ELISA and FACS assays, with no wild-type EGFR binding observed, up to the maximally evaluated TandAb concentration of 1 μM. The most potent TandAbs displayed cytotoxicity towards EGFRvIII expressing F98 glioma and CHO cells with EC50 = 25 pM. We also assayed the cytotoxicity of these TandAbs towards EGFR+ cells as a more sensitive probe of residual binding to the native form. No cytotoxicity was observed up to the maximally evaluated TandAb concentration of 0.5 μM. The cytotoxic potency of the EGFRvIII/CD3 TandAb was higher than that of a comparator bivalent bispecific antibody, constructed from different anti-EGFRvIII/CD3 moieties or of EGFRvIII/CD16A TandAbs, which recruit NK cells instead of cytotoxic T cells. EGFRvIII/CD3 TandAbs with high affinity binding to CD3 were most efficacious for T cell recruitment and tumor cell killing and yet, in the absence of EGFRvIII+ target cells in vitro, TandAbs did not elicit T cell activation, as measured by their lack of proliferation, contributing to a good preclinical safety profile. In vivo EGFRvIII/CD3 TandAbs demonstrated a robust dose-dependent growth retardation of EGFRvIII+ subcutaneous Xenograft tumors in NOD/scid mice reconstituted with human PBMC; this tumor growth inhibition was more pronounced than that associated with cetuximab. In summary, our data demonstrate the strict specificity and high potency of the anti-tumor cytotoxicity mediated by the EGFRvIII/CD3 TandAb, a novel highly potent drug candidate for the treatment of EGFRvIII+ malignancies. Citation Format: Eugene A. Zhukovsky, Kristina Ellwanger, Uwe Reusch, Markus Eser, Fionnuala McAleese, Ivica Fucek, Carmen Burkhardt, Stefan Knackmuss. A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2643. doi:10.1158/1538-7445.AM2014-2643

Abstract C163: A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers.

Abstract To harness the cytotoxic capacity of T cells for the treatment of EGFRvIII+ cancers, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 and EGFRvIII, the EGFRvIII/CD3 RECRUIT-TandAb. EGFR dysregulation has been linked to numerous cancers, and both small molecules and EGFR targeting antibodies have successfully reached the clinic. However, the antibodies approved for clinical use, and those in development, all share a severe side effect profile due to the broad normal tissue-expression of EGFR. The deletion variant III of EGFR (EGFRvIII), with truncated extracellular domain and ensuing ligand-independent constitutive activity, is the most common mutant form associated with oncogenic transformation. EGFRvIII is expressed exclusively in cancer tissues and is associated with various solid tumor types. The restricted EGFRvIII expression on cancer cells provides an opportunity to develop cytotoxic antibodies that solely target cancer, sparing normal tissues, and substantially reduce the side effects associated with EGFR therapy. Using phage display libraries, we identified anti-EGFRvIII antibodies that selectively bound to the mutated and not the native form of EGFR. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies; hence we engineered a panel of bispecific EGFRvIII/CD3 TandAbs, capable of T cell-recruitment, with a broad range of binding and cytotoxic properties. The TandAbs’ binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. They exhibited exquisite specificity towards EGFRvIII in ELISA and FACS assays, with no wild-type EGFR binding observed, up to the maximally evaluated TandAb concentration of 1 μM. The most potent TandAbs displayed cytotoxicity towards EGFRvIII expressing F98 glioma and CHO cells with EC50 = 25 pM. We also assayed the cytotoxicity of these TandAbs towards EGFR+ cells as a more sensitive probe of residual binding to the native form. No cytotoxicity was observed up to the maximally evaluated TandAb concentration of 0.5 μM. The cytotoxic potency of the EGFRvIII/CD3 TandAb was higher than that of a comparator bivalent bispecific antibody, constructed from different anti-EGFRvIII/CD3 moieties; moreover, the comparator was not strictly specific for EGFRvIII and had residual cytotoxicity, EC50 = 1 nM, towards EGFR+ cell lines. High affinity binding to CD3 was essential for efficacious T cell recruitment and yet, in the absence of EGFRvIII+ target cells in vitro, TandAbs did not elicit T cell activation, as measured by their lack of proliferation, contributing to a good preclinical safety profile. In summary, our data demonstrate the strict specificity and high potency of the anti-tumor cytotoxicity mediated by the EGFRvIII/CD3 TandAb, a novel highly potent drug candidate for the treatment of EGFRvIII+ malignancies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C163. Citation Format: Eugene A. Zhukovsky, Kristina Ellwanger, Uwe Reusch, Markus Eser, Ivica Fucek, Carmen Burkhardt. A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C163.

Quantification of cell surface proteins with bispecific antibodies.

Flow cytometry is an established method for fast and accurate quantitation of cellular protein levels and requires fluorescently labeled antibodies as well as calibration standards. A critical step for quantitation remains the production of suitable detection antibodies with a precisely defined ratio of antigen-binding sites to fluorophores. Problems often arise as a consequence of inefficient and unspecific labeling which can influence antibody properties. In addition, the number of incorporated fluorophores necessitates a special normalization step for quantitation. To address these problems, we constructed different mono- and bivalent bispecific antibodies with binding site(s) for the cell surface antigens, cMET, EGFR1/HER1, ErbB2/HER2 or ErbB3/HER3 and with an additional digoxigenin-binding single-chain Fv fusion. The fluorophore Cy5 was covalently coupled to digoxigenin and quantitatively bound by the bispecific antibody. A panel of tumor cell lines was assessed under different culture conditions for absolute receptor expression levels of the indicated antigens and the data were set in relation to mRNA, gene count and immunoblot data. We could reproducibly quantify these receptors, omit the otherwise required normalization step and demonstrate the superiority of a 1 + 1 bispecific antibody. The same antibodies were also used to quantify the number of proteins in intracellular vesicles in confocal microscopy. The antibodies can be stored like regular antibodies and can be coupled with different digoxigenin-labeled fluorophores which makes them excellent tools for FACS and imaging-based experiments.

Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics

To increase the valency, stability and therapeutic potential of bispecific antibodies, we designed a novel recombinant molecule that is bispecific and tetravalent. It was constructed by linking four antibody variable domains (V H and V L) with specificities for human CD3 (T cell antigen) or CD19 (B cell marker) into a single chain construct. After expression in Escherichia coli, intramolecularly folded bivalent bispecific antibodies with a mass of 57 kDa (single chain diabodies) and tetravalent bispecific dimers with a molecular mass of 114 kDa (tandem diabodies) could be isolated from the soluble periplasmic extracts. The relative amount of tandem diabodies proved to be dependent on the length of the linker in the middle of the chain and bacterial growth conditions. Compared to a previously constructed heterodimeric CD3 × CD19 diabody, the tandem diabodies exhibited a higher apparent affinity and slower dissociation from both CD3 + and CD19 + cells. They were also more effective than diabodies in inducing T cell proliferation in the presence of tumor cells and in inducing the lysis of CD19 + cells in the presence of activated human PBL. Incubated in human serum at 37 °C, the tandem diabody retained 90 % of its antigen binding activity after 24 hours and 40 % after one week. In vivo experiments indicated a higher stability and longer blood retention of tandem diabodies compared to single chain Fv fragments and diabodies, properties that are particularly important for potential clinical applications.

Chemical synthesis of bispecific monoclonal antibodies: potential advantages in immunoassay systems

To date, the applications of bispecific antibodies in immunoassay and immunocytochemical procedures have been directed at uniting two different biomolecules through the binding of epitopes on each respective substance. In this study, bispecific antibodies were constructed in which both binding sites were directed to two different epitopes of the same target molecule. Two types of bispecific antibody were constructed; a bivalent bispecific monoclonal antibody and multivalent bispecific polymers. The binding characteristics of each were investigated for changes in specificity and binding strength relative to 1:1 mixtures of parent antibodies. A bivalent bispecific antibody (BBA) was synthesised by the method of Glennie et al. (1985) from monoclonal antibodies recognising the ‘M’ or ‘B’ units of creating kinase (CKMB). The BBA had enhanced specificity for CKMB with diminished recognition of CKMM and CKBB. A less tedious method of producing bispecific antibody involving heterobifunctional cross-linkage was used to produce multivalent bispecific antibodies (MBAs). Certain MBAs constructed to bind ‘M’ and ‘B’ units of CKMB demonstrated enhanced specificity and affinity for CKMB. MBAs were also produced to opposite ends of the 39 amino acid peptide adrenocorticotrophic hormone (ACTH). One of these demonstrated an enhanced affinity of 41-fold. We conclude that while conventional synthesis of bispecific bivalent antibodies is not a practical proposition for immunoassay development, antibodies with similar advantages can be produced with a simple method using the heterobifunctional cross-linker. The production of certain bispecific antibody combinations appears to enhance the formation of antibody-antigen matrices conferring higher binding affinities than can be achieved with an antibody mixture alone.